Cardiovascular disease remains a major health problem throughout the developed world, annually ranking at or near the top in terms of human cost in poor health or death, and in terms of financial cost for treatment and prevention. Despite enormous efforts on the parts of both medical practitioners and basic researchers in human health and related fields, cardiovascular disease continues to be a significant problem. The prevalence and persistence of this medical ill has led to steadily increasing efforts to combat it, including molecular biological investigations of the physiological events attending cardiovascular health and disease.
Actin-activated myosin ATPase (i.e., the actomyosin ATPase) powers muscle contraction in a process regulated by Ca2+ binding to the thin filament-associated proteins, tropomyosin and the troponin complex. The current model for striated muscle (i.e., cardiac and skeletal muscle) contraction has contraction initiated by a rise in the cytoplasmic calcium concentration [Ca2+], which results in binding of Ca2+ to troponin C (TnC). Ca2+-TnC binding induces a series of allosteric changes in TnC, TnI, TnT, the three subunits of troponin, and tropomyosin. These conformational changes allow the myosin head to form a strong cross-bridge with the actin filament. This interaction activates the myosin ATPase, displacing the thin filaments relative to the thick filaments, thus leading to a shortening of the sarcomere and contraction of the muscle.
Cardiac and skeletal muscle contraction is activated by Ca2+ via troponin-tropomyosin in the actin thin filament regulatory system (1-3). Troponin C (TnC) is the Ca2+-binding subunit, troponin T (TnT) is the tropomyosin-binding subunit, and troponin I (TnI) is the inhibitory subunit. Troponin T (1) is the anchoring subunit of the troponin complex (4). Three muscle type-specific TnT isoform genes have evolved in higher vertebrates (5-7) and alternative RNA splicing further produces multiple protein isoforms (8-10). The various TnT isoforms mainly differ in their NH2-terminal structures. The amino acid sequence of the NH2-terminal region of TnT is hypervariable among the cardiac, slow and fast skeletal muscle TnT isoforms and is regulated by alternative splicing during perinatal heart and muscle development (8, 9).
The NH2-terminal region of TnT does not contain any known binding sites for other thin filament proteins (11-13). Nonetheless, deletion of the NH2-terminal cTnT fragment decreases contractility of the heart (14, 16). Deleting the NH2-terminal variable region does not diminish the regulatory activity of troponin (14-16), suggesting that the NH2-terminal variable region of TnT may function as a modulatory or regulatory structure. Supporting this view are alterations in the TnT NH2-terminal structure that affect the Ca2+-regulation of muscle contraction. It has been reported that NH2-terminal alternatively spliced TnT isoforms convey significant changes in the activation of actomyosin ATPase (17). Aberrant splicing of cardiac TnT (cTnT) in the NH2-terminal region is found in both hypertrophic and failing human hearts (18) and in animal models with dilated cardiomyopathy (19, 20). Consistent with the functional effects, studies showed that NH2-terminal alterations in TnT alter the overall protein conformation (21, 22), and the binding of TnT to tropomyosin, TnI and TnC (21, 23).
Serum cardiac troponin T has been used in the diagnosis of acute myocardial infarction for some time. Commercially available assays for serum cTnT, however, have two intrinsic problems. First, there are conserved, or similar, regions common to cardiac and skeletal muscle TnTs, and any assay dependent on the conserved region of cTnT will be compromised by detection of skeletal TnTs, resulting in false positives and reduced measurement accuracy. Second, commercially available diagnostic kits provide materials for immunoassays that rely on antibodies raised against intact, full-length cTnT, an environment in which the highly cardiac-specific N-terminal variable region of full-length cTnT lacks significant immunogenicity.
Like the TnT subunit, the TnI subunit shows a core structure conserved in all TnI isoforms. Cardiac TnI (cTnI) has an approximately 30-amino-acid N-terminal extension that is not present in fast and slow skeletal muscle TnIs. This N-terminal extension does not contain binding sites for other thin filament proteins, but contains serine residues 23 and 24 which are protein kinase A (PKA) substrates. With β-adrenergic stimulation, phosphorylation of these serine residues facilitates myocardial relaxation by decreasing the affinity of TnC for Ca2+.
Notwithstanding this understanding of molecular events implicated in cardiovascular disease, and despite recognition that troponin measurements are central to the diagnosis, management and risk stratification of acute cardiovascular events, existing troponin assays have proven inaccurate and insufficiently reliable. For example, a comparison of the measured troponin found in blood samples using three troponin I assays (Centaur, Architect and point-of-care iSTAT) and one troponin T assay (Roche Elecys) revealed significant discrepancies in the measured quantities of troponin. Jossi et al., Intern. Med. J. 36:326-327 (2006).
Thus, there remains a need in the art for methods of preventing, diagnosing and treating cardiovascular diseases and disorders, as well as methods of screening for therapeutics useful in such methods.